Volume 115, Issue 5, Pages 591-602 (November 2003) Local, Efflux-Dependent Auxin Gradients as a Common Module for Plant Organ Formation  Eva Benková, Marta Michniewicz, Michael Sauer, Thomas Teichmann, Daniela Seifertová, Gerd Jürgens, Jiřı́ Friml  Cell  Volume 115, Issue 5, Pages 591-602 (November 2003) DOI: 10.1016/S0092-8674(03)00924-3

Figure 1 Distribution of Auxin and Its Response in Lateral Root Primordia (A) Spatial pattern of DR5::GUS expression during primordium development: the DR5 activity gradient with the maximum at the primordium tip is gradually established. OL and IL, outer and inner layers; central cells outlined. (B) DR5::GUS in columella initial region of the mature lateral root. (C) 2,4-D treatment: loss of DR5 gradient and induction of DR5 activity in the entire primordium. (D) NPA treatment: impaired DR5 gradient after inhibition of auxin efflux. (E) Immunolocalization of IAA during primordium development: the IAA gradient with the maximum at the tip is gradually established. Inset shows increased signal, however no change of the gradient after the IAA treatment. (F) IAA accumulation in columella initial region of the mature lateral root. Inset shows negative control, no signal without IAA fixation. Division planes marked by arrowheads and developmental stages indicated in the upper right corner: I, II, III, IV, V, and e – emergence. GUS signal in blue (A–D), IAA signal in brown (E and F). Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)

Figure 2 PIN Expression and Localization Patterns during Lateral Root Formation (A) PIN1::GUS expression in derivatives of inner cells. Arrowhead marks the border between inner and outer cells. (B) PIN2::GUS in outer cells. (C) PIN2 polar localization pointing away from the tip (arrows). (D) PIN3::GUS at the base and later appearing in the forming meristem (arrowhead). (E) PIN3 localization in the columella precursors of forming lateral root meristem. (F) PIN4::GUS at the base, in the margins, and later in the meristem (arrowhead). (G) PIN4 localization in margins and the meristem. (H) PIN6::GUS in margins. (I) PIN6::GUS, face view. Developmental stages are indicated in the upper right corner. GUS staining in blue (A, B, D, F, H, and I), protein localization signal in red (C and G) or green (E), nuclear counterstain blue (C and G). Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)

Figure 3 In Vivo Monitored Correlation between Individual Primordium Development and the DR5 Gradient (A and B) Wild-type (A) and pin2 (B) primordium development with correctly established DR5 gradient. (C) Retarded pin1 primordium development without established DR5 gradient. (D) Retarded 35S::PIN1 primordium development, no DR5 gradient. (E) Postembryonic phenotype of pin1 pin3 pin4 pin7 mutant: very short root and cotyledon defects. (F–H) Induction of lateral root development by NAA treatment. Normal primordia development in wild-type (F) seedlings. Affected primordia development in pin3 pin7 (G) and pin1 pin3 pin4 (H) mutants. Numbers indicate time points during primordium development (A–D) or period of NAA treatment (F–H). GUS staining in blue (A–D). Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)

Figure 4 Correlation of PIN1 Relocation, DR5 Gradients, and Primordium Development (A and B) PIN1:GFP (A) and PIN1 (B) localization in developing lateral root primordium with gradual establishment of PIN1 at the lateral cell membranes. (C) Nonpolar PIN1 localization in all cells of 35S::PIN1 lateral root primordium. (D–F) PIN1:GFP rearrangement in primordium initiated after IAA treatment (D), no relocation to lateral sides after IAA + BFA (E) or IAA + NPA treatments (F). (G–O) Misexpression of molecular markers after inhibition of PIN1 relocation. Left image after treatment with IAA, right image after IAA + NPA (n) or BFA (b). Auxin response: DR5rev::PEH A (G); cell division: CycB1:GUS (H); margins: CUC3::GUS (I); PIN6::GUS (J) and M0223 (K); inner layer: Q0990 (L); pericycle (arrowheads): M0171 (M); columella and protophloem: AUX1::GUS (N); stele of the primary root: Q0680 (O). Arrowheads indicate PIN1 polar localization (A,B, D–F). GUS and PEH A signal in blue (G–J and N), GFP signal in green (A, D–F, K–M, and O), PIN1 signal in yellow (B) or red (C), nuclear counterstain blue (C). Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)

Figure 5 PIN-Dependent Auxin Gradients in Cotyledon Formation (A) DR5rev::GFP in triangular embryo. Signals (arrowheads) visible in the basal region and in the tips of incipient cotyledons. (B) IAA immunolocalization mirroring the DR5 pattern in heart stage embryo. (C) Defects in the DR5 gradients and cotyledon formation in the gnom embryo. (D and E) BFA treatment: defects in DR5 gradient and cotyledon formation in control embryo (D). No BFA effects on DR5 gradients and cotyledon formation in BFA resistant GNOMM696L-myc embryo (E). (F and G) Defects in cotyledon formation in pin1 (F) and pin4 pin7 (G) mutants. (H–J) PIN1 localization in embryos. In the globular embryo stage, the basal PIN1 localization in cells below incipient cotyledons is not established yet (H). Gradual establishment of PIN1 basal localization at triangular (I) and early heart (J) stages. (K and L) Schematics of putative PIN1-dependent auxin fluxes (arrows) at globular (K) and triangular (L) stage. (M) NPA treatment prevents the establishment of PIN1 basal localization in provascular cells. (N) PIN1:GFP polarity in the epidermis of heart embryos pointing toward the incipient cotyledon tips. Arrowheads indicate PIN1 polar localization (H–J, M, and N). GFP signals in green (A, C–E, and N). IAA signal in brown (B) and PIN1 signal in red (H–J, and M). Nuclear counterstain in blue (H–J, and M). Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)

Figure 6 PIN-Dependent Auxin Gradients in Shoot-Derived Organ Formation (A and B) DR5rev::GFP in the apical meristem. Accumulation of signals visible at the position of incipient primordia and their tips (A). Auxin efflux inhibition by NPA interferes with the DR5 signal at tips (B). (C and D) DR5rev::GFP in developing flowers. Signals visible at the tips of all floral organ primordia (C). Auxin efflux inhibition by NPA interferes with the DR5 signals at tips (D). (E and F) PIN1 localization in apical (E) and secondary floral (F) meristems. In the incipient vasculature below the primordia, PIN1 basal localization is gradually established (inset in E). PIN1 localizes in the epidermis cells toward primordia tips (inset in F). (G) PIN1:GFP in epidermis and incipient vasculature in developing flowers. (H) PIN1:GFP polar localization (inset) pointing toward the tips of floral organ primordia. (I) Pin-like inflorescence in wild-type plant grown on NPA. (J) pin1 inflorescence without the initiated lateral organs. (K) Defective flowers in pin3 pin7 mutants. Missing stamens and carpels indicated (arrowheads). Inset shows example with missing sepals, petals, and fused petals. (L and M) Young ovule primordium. DR5rev::GFP signal at the tip (L). PIN1:GFP polar localization in outer layer (M). (N and O) Older ovule primordium. DR5rev::GFP signal at the tip and at tips of developing integuments (arrowheads) (N). PIN1:GFP polar localization in outer layer and in inner cells (O). (P) PIN1 signals in developing ovule primordia in wild-type gynoecium. (Q) pin1 gynoecium, no or entirely misshaped (arrowhead) ovules develop. Arrowheads mark incipient primordia and their tips (A–E, and N) or PIN1 polar localization (F, H, and M). GFP signal in green (A–D, F–H, and L–O) and PIN1 signal in yellow (E and P). Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)

Figure 7 Model for Auxin Transport and Distribution in Root- and Shoot-Derived Organ Primordia (A) Lateral root primordium: auxin is provided by PIN-dependent auxin transport through the primordium interior toward the tip, where it accumulates. From here, part of the auxin is retrieved by a PIN2-dependent auxin route through the outer layers. (B) Aerial organ primordium: PIN1 is a major component for auxin distribution. Auxin is provided to the primordium tip through the outer layers. From the tip, auxin is drained through a gradually established transport route toward the vasculature. Places of auxin accumulation are depicted in green. Presumptive routes of auxin transport are depicted by red arrows. Cell 2003 115, 591-602DOI: (10.1016/S0092-8674(03)00924-3)